菊花淹水响应基因CmRAP2.3的克隆与功能分析

摘要：菊花(Chrysanthemum morifolium)是我国十大传统名花之一，栽培历史悠久，在园林观赏和绿化中具有极高的价值。菊花生性强健，但是耐涝性差，常常在生产上或园林规划运用中造成严重的损失。本论文旨在研究菊花淹水响应基因CmRAP2.3的基因序列及其功能，从而初步阐明菊花的淹水胁迫响应机制。本研究以菊花切花品种‘南农雪峰’为实验材料，提取总RNA并反转录cDNA，通过高保真PCR获得CmRAP2.3基因的全长序列；采用实时荧光定量PCR的分析方法，研究了CmRAP2.3基因在淹水环境下的表达，通过酵母单杂交验证了CmRAP2.3基因的转录激活活性，并且利用洋葱表皮细胞瞬时表达系统对该基因进行了亚细胞定位。结果表明，CmRAP2.3基因的开放阅读框为702bp，编码234个氨基酸，并且具有典型的ERF类转录因子家族的DNA结构域。实时荧光定量PCR分析表明，CmRAP2.3基因在淹水条件下出现过量表达。酵母单杂交实验表明该基因具有转录激活活性。亚细胞定位证明该基因定位于细胞核中。初步推断，CmRAP2.3基因参与了植物淹水胁迫的响应过程，并且对植物在淹水环境下的调节起到重要的作用。30133
毕业论文关键词：淹水胁迫；菊花；ERF类转录因子；基因克隆
CLONING AND FUNCTIONAL VERIFICATION OF RAP2.3 GENE IN CHRYSANTHEMUM
Abstract: Chrysanthemum is one of Chinese top ten traditional flowers, which has a long history of cultivation. It has a very high value in garden ornamental enjoying and greening. Chrysanthemum is very strong, but its flooding tolerance is poor, which often causes serious losses in production or landscape planning. The aim of this thesis was to study on the gene sequence and function of CmRAP2.3 gene in chrysanthemum and explained the response mechanism to flooding stress in chrysanthemum. In this study, we used cut flower variety ‘NanNongXueFeng’ as the experimental materials. We extracted the total RNA and obtained the reverse transcription cDNA , the full length of CmRAP2.3 gene was also obtained by high-fidelity PCR. The real-time quantitative PCR method was used to study the expression of CmRAP2.3 gene in the flooded environment. Furthermore, the transcriptional activation of CmRAP2.3 gene was verified by the yeast one-hybrid assay and its subcellular localization was analyzed by transient expression in onion epidermal cells. The results showed that the open reading frame of CmRAP2.3 gene was 702 bp, which encoded 234 amino acids. This gene had a typical DNA domain of the ERF transcription factors. The real-time quantitative PCR showed that the CmRAP2.3 gene was over-expressed under the flooding condition. The yeast one-hybrid assay showed that the CmRAP2.3 gene had the transcriptional activation function. The subcellular localization demonstrated that the gene was located in the nucleus. It can be concluded that CmRAP2.3 gene involves the process of plant’s response under the flooding stress and plays an important role in regulation of plant which is in the flooded environment.
Key words: flooding stress; Chrysanthemum morifolium; ERF transcription factor; gene cloning
目录

摘要    4
关键词    4
Abstract    4
Key words    4
第一章引言    5
第二章 CmRAP2.3基因克隆与序列分析    6
1 材料与方法    6
1．1 植物材料及处理    6
1．1．1 植物材料    6
1．1．2 处理    6
1．2 实验方法    6
1．2．1 总RNA提取    6
1．2．2 第一链cDNA的合成    6
1．2．3 高保真PCR扩增    7
1．2．4 目的片段连接    8 菊花淹水响应基因CmRAP2.3的克隆与功能分析:http://www.youerw.com/shengwu/lunwen_25686.html